Abstract The Gram-negative sub-gingival anaerobic bacterium Porphyromonas gingivalis is closely associated with periodontitis, a chronic inflammatory disorder. It is found in greater abundance in periodontal diseased sites than in healthy sites suggesting it is well equipped to survive hostile inflammatory conditions. In addition, mouse models of infection have demonstrated that infection with P. gingivalis can tilt the sub-gingival microbiome to a disease-inducing dysbiotic state. Lipid A, the hydrophobic anchor of lipopolysaccaharide, is the bacterial ligand of the host innate immune receptor, TLR4. In P. gingivalis, lipid A is modified by deacylation, from an initially synthesized penta- acylated structure to tetra-acylated structures, a step essential for evading the TLR4-mediated pro- inflammatory response. Tetra-acylated lipid A is also known to contribute significantly to bacterial virulence properties such as outer membrane vesicle (OMV) formation, and attenuation of inflammasome activation. OMV's in P. gingivalis are distinct from the outer membrane (OM) in that they contain more under- acylated lipid A, more Type IX secreted proteins (T9SS), and more anionic A-LPS, the last known to be an OM tether for T9SS proteins. T9SS proteins comprise many virulence proteins including gingipain proteases and peptidylarginine deiminase. OMVs, hence, are considered as long distance carriers of a heavy virulence payload. Since lipid A molecules of OMVs have been shown to be tetra-acylated, and OMVs are a favored environment for T9SS virulence proteins, we hypothesize that the OM of a deacylase deficient strain is, conversely, attenuated for these virulence factors due to absence of tetra-acylated lipid A. Identification of the as yet unknown lipid A deacylase, required for a key step in the lipid A biology of P. gingivalis, was the focus of our previous R21 application. Our work led to identification of PGN_1123, a highly conserved gene, as the deacylase-encoding gene in P. gingivalis 33277. In this application, our goal is to study the effect of lipid A deacylation on different aspects of P. gingivalis pathogenesis. In Aim 1, we will compare wild-type 33277 with the ?PGN_1123 mutant for OMV production, amount of A-LPS, gingipain activity and inflammasome activation, all of which contribute to P. gingivalis virulence. In Aim 2, we will investigate whether expression of PGN_1123 is up-regulated during disease. PGN_1123 is the last gene of a three gene operon. We will compare in vivo expression of all three genes of the operon from healthy vs. diseased sub-gingival plaque samples obtained from human subjects. A decrease in potency of virulence properties in the ?PGN_1123 mutant will be followed by an in depth study of the enzyme to better understand the deacylation process, and to devise ways of inhibiting it. Increased PGN_1123 expression during disease will imply the gene is regulated. Future studies will include investigating its regulation, and evaluating use of its expression as a diagnostic tool for measuring extent of disease.